1. Field of the Invention
The present invention relates to arrays of labeled microparticles. Such arrays are particularly useful in multiplex assays, such as biological detection assays and, more particularly, in the fields of flow cytometry and fluorescence microscopy.
2. Description of Related Art
Flow cytometers are well known analytical tools that enable the characterization of particles on the basis of light scatter and particle fluorescence. In a flow cytometer, particles are individually analyzed by exposing each particle to an excitation light, typically one or more lasers, and the light scattering and fluorescence properties of the particles are measured. Particles, such as molecules, analyte-bound beads, individual cells, or subcomponents thereof, typically are labeled with one or more spectrally distinct fluorescent dyes, and detection is carried out using a multiplicity of photodetectors, one for each distinct dye to be detected. Flow cytometers are commercially available from, for example, BD Biosciences (San Jose, Calif.).
Early in the development of flow cytometry, it was recognized that various types of ligand binding assays could be carried out using microparticles (beads) coated with one member of a binding pair. For example, immunoassays can be carried out in a sandwich hybridization assay format using beads coated with an analyte-specific binding agent, such as a monoclonal antibody (mAb), as a capture reagent, and a second analyte-specific binding agent, again typically a mAb, labeled with a fluorophore as a reporter reagent. The coated beads and reporters are incubated with a sample containing (or suspected of containing) the analyte of interest to allow for the formation of bead-analyte-reporter complexes. Analysis by flow cytometry enables both detecting the presence of bead-analyte-reporter complexes and simultaneously measuring the amount of reporter fluorescence associated with the complex as a quantitative measure of the analyte present in the sample.
It was also recognized early in the development of flow cytometry that the simultaneous analysis of multiple analytes in a sample could be carried out using a set of distinguishable beads, each type of bead coated with a unique analyte-specific binding agent. The bead set and fluorescently labeled reporter reagents, one for each species of analyte to be detected, are incubated with a sample containing the analytes of interest to Is allow for the formation of bead-analyte-reporter complexes for each analyte present, and the resulting complexes are analyzed by flow cytometry to identify and, optionally, quantify the analytes present in the sample. Because the identity of the analyte bound to the complex is indicated by the identity of the bead, multiple analytes can be simultaneously detected using the same fluorophore for all reporter reagents. A number of methods of making and using sets of distinguishable microparticles have been described in the literature.
UK Patent No. 1 561 042, published Feb. 13, 1980, and Fulwyler and McHugh, 1990, Methods in Cell Biology 33:613-629, describe the use of multiple microparticles distinguished by size, wherein each size microparticle is coated with a different target-specific antibody.
Tripatzis, European Patent No. 0 126,450, published Nov. 28, 1984 (see also corresponding Canadian Patent 1 248 873), describes multi-dimensional arrays of microparticles formed by labeling microparticles with two or more fluorescent dyes at varying concentrations. Microparticles in the array are uniquely identified by the levels of fluorescence dyes. Tripatzis describes the use of such arrays for the simultaneous detection a large numbers of analytes in a sample by flow cytometry, and, further, describes their use as labels in microscopy.
U.S. Pat. Nos. 4,499,052 and 4,717,655, Entitled: “Method and Apparatus for Distinguishing Multiple Subpopulations of Cells”, issued Feb. 12, 1985, and Jan. 5, 1988, respectively, describe the use of microparticles distinguishably labeled with two different dyes, wherein the microparticles are identified by separately measuring the fluorescence intensity of each of the dyes.
Both one-dimensional and two-dimensional arrays for the simultaneous analysis of multiple analytes by flow cytometry are available commercially. Examples of one-dimensional arrays of singly dyed beads distinguishable by the level of fluorescence intensity include the BD™ Cytometric Bead Array (CBA) (BD Biosciences, San Jose, Calif.) and Cyto-Plex™ Flow Cytometry microspheres (Duke Scientific, Palo Alto, Calif.). An example of a two-dimensional array of beads distinguishable by a combination of fluorescence intensity (five levels) and size (two sizes) is the QuantumPlex™ microspheres (Bangs Laboratories, Fisher, Ind.). An example of a two-dimensional array of doubly-dyed beads distinguishable by the levels of fluorescence of each of the two dyes is described in McDade and Fulton, April 1997, Medical Device & Diagnostic Industry; and Fulton et al., 1997, Clinical Chemistry 43(9):1749-1756.
Each of the microparticle arrays described above has disadvantages that limit their utility. Arrays based on different size microparticles are problematical because the amount of capture reagent that can be bound to a microparticle, which affects the sensitivity and dynamic range of the assay, is dependent on the particle size. Thus, to obtain uniform assay performance for all analytes, it is desirable to use microparticles of uniform size. One-dimensional arrays based on differences in the fluorescent intensity of a single dye typically are limited to about 10 different microparticle populations. Although useful for a wide range of assays, it is desirable to have more distinct microparticle populations to enable the simultaneous detection of greater numbers of analytes. Two-dimensional arrays based on differences in the fluorescence intensities of two distinct dyes enable much larger arrays, but are significantly more difficult to manufacture, and increase the difficulty in subsequent data analysis.